Reoviruses provide a well-established experimental system for studies of viral neuropathogenesis. Following primary infection in the intestine of newborn mice, type 3 reoviruses spread through nerves and infect neurons, causing encephalitis. Viral attachment protein sigma1 plays a crucial role in each of these progressive pathologic events. The sigma1 protein is a fibrous protein consisting of an N-terminal tail and a C-terminal head. The sigma1 tail contains a domain that binds sialic acid; the sigma1 head binds junctional adhesion molecule-1 (JAM-1). Recent high-resolution structural studies indicate that the JAM-1-binding domain of sigma1 is a homotrimer of loosely associated eight-stranded beta-barrels. However, the mechanism of dual receptor binding by sigma1 is not known, nor is it understood how sigma1-receptor interactions dictate viral tropism, cell entry, or disassembly. Four integrated specific aims are proposed to study structural and functional properties of sigma1, with the goal of defining the structural basis for the interaction of sigma1 with its receptors. In Specific Aim 1, the structural basis of sigma1 interactions with JAM-1 and sialic acid will be determined using X-ray crystallography. Truncated fragments of sigma1 that comprise the receptor-binding domains and the extracellular region of JAM-1 will be used for these analyses. In Specific Aim 2, local determinants of sigma1 receptor binding and the global relationships between the sigma1 receptor-binding domains will be defined. The length of a linker sequence between the receptor-binding domains will be altered, and modified sigma1 protein will be tested for viral attachment. In Specific Aim 3, determinants of conformational changes in sigma1 will be identified using X-ray crystallography and circular dichroism. The role of conserved aspartic acid residues at the head trimer interface will be defined using directed mutagenesis and assays of viral attachment and disassembly. In Specific Aim 4, dynamic regions of sigma1 structure will be defined using a very high-resolution structure of the G1 C-terminal domain and molecular dynamics simulation. These studies will yield a precise understanding of reovirus cell-attachment and provide insights into mechanisms by which reovirus selects cellular targets and produces neurologic disease. Moreover, since the atomic structures of so few virus-receptor complexes are known, this work may serve as a general model for ligand-receptor binding and aid in the rational design of antiviral therapeutics based on interference with specific pathogen-receptor interactions.